Lutein is a naturally occurring carotenoid that has no vitamin A activity. There are three asymmetric centers in lutein at C-3, C-3', and C-6' positions. The absolute configuration of lutein in foods (fruits and vegetables) and human serum/plasma is known to be 3R,3'R,6'R. This configurational isomer of lutein which is the most abundant form of lutein is also known as lutein A. However in human serum/plasma a small amount of another configurational isomer of lutein, namely (3R,3'S,6'R)-lutein also known as 3'-epilutein or lutein B has been shown to be present. The chemical structures of lutein and 3'-epilutein are shown below. Other known configurational isomers of lutein have only been isolated from the integument of marine fishes. ##STR1##
The terms all-E- and Z-isomers are new terminology used for lutein and other carotenoids, and refer to old terminology of all-trans and cis isomers of these compounds, respectively. Unless specified lutein refers to all -E (all-trans) isomer.
Numerous epidemiological studies in various populations have shown that the consumption of substantial amounts of fruits and vegetables reduce the risk of cancer. One hypothesis is that the carotenoids in these foods act as antioxidants through a free radical mechanism by quenching singlet oxygen and other oxidizing species resulting in the termination of free radical chain reactions and in the prevention of cellular oxidative damage. The correlation between dietary carotenoids and carotenoids found in human serum or plasma indicate that only selected groups of carotenoids make their way into the human bloodstream to exert their effect. To date, 19 carotenoids have been identified to be present in human blood. With the exception of .beta.-carotene, none of these carotenoids have been studied for their anti-cancer activity.
There has been recent evidence to suggest that lutein, one of the most abundant carotenoids in the diet and in human blood possesses strong antioxidant capabilities and may be useful in reduction of the incidence of cancer. The allylic hydroxyl group at the C-3' position of the E-end group of this compound can readily undergo oxidation as a result of activation by the neighboring double bond. The non-allylic hydroxyl group at the C-3 position of the .beta.-end group can also activate the C-4 carbon allylic to the double bond making this carbon highly susceptible to direct oxidation. These oxidation reactions result in the formation of four metabolites of lutein, which have been isolated and characterized from extracts of human serum or plasma. Further evidence for an in vivo oxidation of lutein has also been obtained in human feeding studies. To date, pure lutein suitable for human use has not been commercially available for use as a chemopreventive agent in clinical trials. Pure lutein, free from chemical contaminants and suitable for human consumption, is needed to design and conduct proper human intervention studies.
Lutein is one of the major constituents of green vegetables and fruits such as broccoli, green beans, green peas, lima beans, Brussels sprouts, cabbage, kale, spinach, lettuce, kiwi, and honeydew. The lutein in these green vegetables and fruits exists naturally in the free non-esterified form and co-exists with other carotenoids.
However, the isolation and purification of free form, pure lutein in large quantities from green vegetables is not economical. Many expensive and time-consuming purification steps are required to remove and purify lutein from the large quantities of chlorophylls, .beta.-carotene, and carotenoids epoxides that are also present in green vegetables.
Lutein is also abundant in a number of yellow/orange fruits and vegetables such as mango, papaya, peaches, prunes, acorn squash, winter squash, and oranges. Lutein in these yellow/orange fruits and vegetables exists in the esterified form with fatty acids such as myristic, lauric, and palmitic acids. Upon ingestion of these foods, lutein esters undergo hydrolysis to regenerate free lutein which is then absorbed and metabolized by the body. However, these yellow/orange fruits and vegetables also contain high concentrations of a number of other carotenoids which make the isolation and purification of lutein costly and time-consuming.
Marigold flower petals are an excellent source of lutein because they contain high levels of lutein and no significant levels of other carotenoids. Extracts of marigold flowers are commercially available but consist of lutein that is esterified with fatty acids such as lauric, myristic, and palmitic acids. Lutein in its natural form as it exists in marigold flowers does not exist as free lutein. Upon saponification of the marigold extract, the lutein fatty acid esters are converted to lutein. However, the resulting lutein is still contaminated with a number of chemical impurities. To date, no method has been described to isolate and purify the free form of lutein from these chemical impurities.
The saponified extracts of marigold flower petals are commercially available and currently used in chicken feed to enhance the yellow color of egg yolk and the skin of chickens. However, the extract is not acceptable as a direct color additive for human foods because of the presence of impurities. The availability of substantially pure lutein suitable for human use and the evidence that significant levels of lutein derivatives are normally found in human blood would also make lutein an attractive color additive.
While chemical processes for synthesis of lutein from commercially available starting materials are known, such processes are extremely time-consuming, involve multiple steps, and to date have simply not provided an economical process for production of lutein. It therefore appears that the most likely economic route to substantially pure lutein is through a process that extracts, isolates, and purifies lutein from marigold flowers. Such substantially pure lutein, if economically available, could be used in cancer prevention studies, as well as an attractive, naturally-occurring, non-harmful color additive in human foods.
It is therefore evident that there is a need for a carotenoid composition of substantially pure lutein. The primary objective of the present invention is the fulfillment of this need.
Another objective of the present invention is to provide a method for isolating, purifying and recrystallizing lutein in high purity from a saponified marigold extract.
Another objective of the present invention is to provide a method for isolating, purifying and recrystallizing lutein in high purity which is economical to perform with a minimum of processing steps.
Another objective of the present invention is to provide purified lutein in crystalline form such that it is acceptable for human consumption and use in cancer prevention trials and treatments without causing toxic side effects due to residual impurities.
Yet a further objective of the present invention is to provide purified lutein in crystalline form such that it is acceptable as a color additive in human foods.
The method and means of accomplishing each of the above objectives as well as others will become apparent from the detailed description of the invention which follows hereinafter.